Immunoassays where one or more antibodies are used to detect a test substance (target, analyte) in a test sample are widely known. A standard application of this technique is the Enzyme Linked Immunosorbent Assay (“ELISA”). The ELISA either uses a capture antibody immobilized on a solid surface for specifically capturing a target antigen from a complex biological matrix (sandwich immunoassay format) or the target antigen to be detected is non-specifically adsorbed to a solid surface, the solid surface typically being the inside of a microtiter plate well. Unbound matrix is removed by a washing step and subsequently followed by coupling of an enzyme-labelled detection antibody (direct ELISA) to the target or coupling of a detection antibody specific for the target antigen to the target followed by coupling of a secondary enzyme-labelled antibody to the primary antibody (indirect ELISA). The enzyme activity associated with the solid surface which is subsequently determined is directly proportional to the amount of bound antigen present and can be measured, for example, by using a chromogenic substrate for the enzyme.
The evolution of immunoassay methods increased the sensitivity of these tests by altering the detection principle. In the course of this development, the enzyme-coupled detection antibody was replaced by oligonucleotide (e.g. DNA) labelled antibodies. In these antibody-nucleic acid conjugates the oligonucleotide served as a marker that could be subsequently amplified and detected. In an application of the PCR (“polymerase chain reaction”) as an exponential amplification system for nucleic acids to these antibody-based detection system (Sano et al. (2000), “Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates” Science 258(5079):120-122), the Immuno-PCR (IPCR) method was developed. The efficacy of this method was first demonstrated for the detection of Bovine Serum Albumin (BSA) as an antigen passively absorbed to an immuno-assay plate. Using an antibody specific for BSA coupled to a biotin-labeled reporter DNA plasmid by means of a protein A-avidin fusion protein, and subsequently utilizing 30 cycles of polymerase chain reaction (PCR) amplification to amplify the reporter DNA sequence, the detection of the amplicons by staining with ethidium bromide following gel electrophoresis was possible. Sano et al. (supra) reported an enhanced detection sensitivity of approximately five orders of magnitude when compared to ELISA detection. Theoretically, using this technique, a nucleic acid label when amplified by PCR or any other available exponential nucleic acid amplification technique can be detected with extraordinary sensitivity (potentially down to a single copy).
However, because of the liability of the protein A-avidin fusion protein to bind any antibody present, e.g. the capture antibody in a sandwich immunoassay, other means of attaching the DNA to the reporter antibody had to be found.
This was achieved by substituting the protein A-streptavidin fusion protein with a biotinylated detection antibody coupled to biotinylated DNA via sequential incubation of the antibody and the DNA with streptavidin as a tetravalent biotin-binding linker molecule (Zhou et al. (1993). “Universal Immuno-PCR for ultra-sensitive target protein detection.” Nucleic Acids Res 21(25): 6038-9) or direct conjugates synthesized by covalently coupling antibodies and DNA (Hendrickson et al. (1995). “High Sensitivity Multianalyte Immunoassay Using Covalent DNA-Labeled Antibodies and Polymerase Chain Reaction.” Nucleic Acids Res. 23(3): 522-529). With these strategies, a sandwich IPCR using antigen-specific capture and detection antibodies which was similar to conventional ELISA analysis and therefore able to detect antigens in complex biological matrices became accessible (Maia et al. (1995). “Development of a two-site immuno-PCR assay for hepatitis B surface antigen.” J. Virol. Methods 52(3): 273-86).
Immuno-PCR is nowadays used in combination with several matrices for the detection of a number of different antigens, including virus particles, tumor markers or cytokines in various body fluids (Niemeyer et al. (2005) “Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification.” Trends Biotechnol. 23(4): 208-16).
Due to the enormous exponential signal amplification, however, Immuno-PCR, as used in state of the art applications, is very susceptible to background effects by unspecific binding of sample contents and the involved reagents, especially during the antigen binding step. Since theoretically a single molecule of nucleic acid can be detected by PCR, failure to remove all of the non-specifically bound template DNA results in significant background compared to conventional ELISA techniques and interferes with the ability to detect minute quantities of analyte, because the signals from these unspecific binding events are amplified in IPCR, too. As well known among scientists working in this field, a background signal is, in contrast to other standard PCR techniques where signals for the negative control are typically not detectable, measured in all typical IPCR assays.
The art of improving IPCR assays is thereby mainly an attempt to either decrease the unavoidable background signal or to increase specific signals induced by the analyte in order to improve the signal-to-background ratio.
Niemeyer et al. (Niemeyer et al. (1999), Nucleic Acid Research 27(23): 4553-4561) have reported that the use of supramolecular antibody-modified DNA-STV oligomers as detection reagents in IPCR led to an enhanced sensitivity compared to the conventional IPCR procedure.
Nevertheless, there remains a need in the field of diagnostics and biosciences for strategies to further improve IPCR assay performance. Thus, one object of the inventors of the present invention was to provide an IPCR assay with further improved assay performance compared to known assays.